The use of reversed cubic and liquid crystalline phases in the field of controlled release devices is described in EPO 125 751 (Engstrom et al 1983). However, there are many applications where the use of a homogeneous phase is not manageable. By the present invention it has become possible to prepare particles, especially colloidal particles, of such phases or similar phases, viz by means of a new fragmentation technique. The importance of said new technique, and the new particles obtained thereby, can be better understood from the following brief review of the prior art concerning other Types of particles and homogeneous phases.
2.1. Dispersed lipid-based systems in pharmaceutical preparations
Essentially, there have To date been three major particulate colloidal lipid-water systems which have been considered as suitable for drug delivery, namely such based on the lamellar mesophase as liposomes, micellar-based phases including micelles, reversed micelles, and mixed micelles and various kinds of emulsions including microemulsions, as well as more novel carriers as ISCOM's (Morein 1988) (a general text concerning these systems is Pharmaceutical dosage forms, Disperse systems 1988). The latter system has been utilized for intravenous nutrition since the beginning of this century and as an adjuvant system known as the Freunds adjuvant. These are of oil-in-water (O/W) and water-in-oil (W/O) types, respectively. Liposomes have since their discovery been extensively investigated as drug delivery systems for various routes and drugs. The development of new colloidal drug carrier systems is a research area of intensive activity and it is likely that new systems, especially new emulsion based systems, will appear in the near future (cf. Weiner 1990a, 1990b). Lipid-based vehicles can take several different morphological forms such as normal and reversed micelles, microemulsions, liposomes including variants as unilamellar, multilamellar, etc., emulsions including various types as oil-in-water, water-in-oil, multiple emulsions, etc., suspensions, and solid crystalline. In addition so called niosomes formed from nonionic surfactants have been investigated as a drug vehicle. The use of these vehicles in the field of drug delivery and biotechnology is well documented (Mulley 1974, Davis et al. 1983, Gregoriadis 1988a, Liebermann et al 1989). Particularly in the field of drug delivery the use of lipid-based drug delivery systems, especially dispersed systems, has attained increasing interest as the pharmaceutical industry is developing more potent and specific--and thus more (cyto)toxic--drugs. This is because the vehicle can in principle reduce such toxic effects and/or side-effects, due to sustained release or increased site-specificity. The current invention is easily distinguished from these earlier lipid-water based systems, as follows:
The term liposomes is conceptually wrong in view of the current knowledge of polymorphism of lipids. Liposome means "lipid body" and has by many authorities in the field been defined as any structure with an enclosed volume that is composed of lipid bilayers (see eg. Tice and Tabibi 1992). This is not only very misleading but also conceptually wrong. Such a definition means that any dispersed lipid based structure built up by a bilayer should fall into this category of device without regarding the different crystallographic aspect of the undispersed, homogeneous, phase from which the particulate vehicle is derived. It would, however, not include dispersions in which the interior of the particles is made up by reversed hexagonal phases, since they are built up by a monolayer, rather than a bilayer. Unfortunately, the concepts of lipid polymorphism and in particular the more complex structures of cubic liquid crystalline phases are often overlooked. Since the current disclosure is in the field of lipid-based vehicles, in which various reversed lyotropic liquid crystalline phases are enclosed in a volume whose boundary is made up by L3 phase or lamellar crystalline phase or lamellar liquid crystalline phase, or a combination thereof, it should be stressed that the current invention encloses either lipid bilayer or monolayer structures different from the lamellar phase. The ordered interior of each particle in the current invention is a portion of a lipid-water microstructured phase that is a thermodynamically stable phase, either a cubic, hexagonal, intermediate phase or an L3 phase. The L3 phase is not classified as a liquid crystalline phase, as the others, rather it is an isotropic solution phase, using the standard nomenclature in the literature of amphiphile microstructures. The physical properties of the homogeneous reversed liquid crystalline phases used in the currect disclosure are those presented in the patent by Engstrom et al. (1983) referred to. In the cases where a cubic phase constitutes the interior of the particles it is built up by a bi- or multicontinuous interpenetrating network microstructure, at the scale of nanometers. This makes these phases unique with regard to compartmentalization since the two independent interpenetrating networks separated by the bilayer can be distinguished, and endows them with extremely high specific surface area, which is especially important in the formulation of amphiphilic drugs straddle hydrophobic and hydrophilic microdomains.
The current invention is thus easily and sharply distinguished from both liposomes, emulsions, microemulsions, as well as various microencapsulated emulsions, hydrogels, and reversed micelles. Most obviously, the interior phase(s) of the current particles is (are) a thermodynamic equilibrium phase, and thus appears as a discrete region in a phase diagram which obeys the phase rule of Gibb's and other laws of chemical and thermodynamical equilibria; this is in sharp contrast with liposomes and emulsions, which are non-equilibrium states or morphologies. [Note that we are using the convention of referring to equilibrium structures as "phases" and non-equilibrium structures as "states"]. In the case of emulsions the interior is also thermodynamical stable, but it is an interior which lacks long-range order, and is not composed of either lipid bilayers or lipid monolayers, or analogous structural elements. This is a clear distinction, which is directly accessible to experiment, since the interior phase used in the current particles give rise to Bragg peaks on examination with small-angle X-ray (or neutron) scattering techniques, in accordance with its lattice ordering; thus the Bragg peaks recorded can be indexed to e.g. a simple cubic, body-centered cubic, or face-centered cubic lattice, hexagonal lattice, or tetragonal lattice in the case the interior is made up by a cubic phase, a hexagonal phase or intermediate tetragonal phase, respectively. In contrast, no case has ever been reported in which multiple Bragg peaks, indexing to any of these lattices, were recorded in a small-angle scattering experiment on a liposomal dispersion or an emulsion. Clearly, the surface of the current particles can in practice give rise to diffraction indexing on a lamellar lattice. In the case of microemulsions and reversed micellar phase, both lacking long range order, they are clearly distinguished from the L3 phases in the current surfactant literature.
The distinctions between the current invention on the one hand, and the liposomal dispersions and emulsions on the other, then follow directly from the above distinctions, and it is only in the case of the reversed liquid crystalline phase dispersions disclosed herein the interiors of the particles are substantially composed of regions of reversed liquid crystalline phase(s).
In the case of dispersions of L3 phases, the interiors of the particles are not composed of liquid crystalline material but of the to the cubic phases closely related L3 phases. The L3 phases are thermodynamic equilibrium phases, distinguishing them from liposomes and emulsions as in the case of cubic phases. The lipid film forms a highly connected bilayer as in the cubic phase, again in contrast with the liposomes and emulsions. However, in this case scattering experiments do not reveal long-range order as in the cubic phase.
Of special importance in formulations, used either for drug delivery or for biological or biotechnological applications, is the position and orientation of the compound with respect to-the bilayer. In the current invention specific orientation may be readily achieved in the case the interior is composed of cubic phase, for which it is an inherent property, as opposed to liposomal bilyaers. This substantially simplifies the process of standardizing enzyme activity in the formulation. Such selectivity in membrane topography is not easily established in other lipid-based systems such as liposomes and emulsions. There are several other areas of interest where the presented topography of the compound is of profound importance, as with antigen presentation in immunization processes. The current invention can accomplish the optimization of this presentation for both extracellular and intracellular targets.
2.2. Homogeneous liquid crystalline phases in pharmaceutical preparations
Liquid crystals do participate in the microstructure of pharmaceutical preparations, and probably do so more frequently than is usually expected. The use of homogeneous reversed cubic and hexagonal phases as a controlled release system for use in e.g. drug delivery systems was invented by Engstrom, Larsson, and Lindman in Lund, Sweden, who are holders of a current patent (Engstrom et al. 1983, see also Ericsson et al 1991, and references therein). Dr. D. Attwood and coworkers in Manchester, UK, have also investigated the use of cubic phases for the purpose of drug delivery (cf. Burrows et al 1990).
Cf. also Mueller-Goymann and collaborators (Mueller-Goymann 1985, Mueller-Goymann 1987, Mueller-Goymann 1989 and references within these works). Other contributions occur in the literature (cf. Ibrahim 1989, Tyle 1990) and are not restricted to lyotropic liquid crystals (Loth and Euschen 1990).
2.3. Dispersed reversed cubic liquid crystalline phases
There have been speculations of the existance of dispersed cubic liquid crystalline phases in connection with fat digestion (cf. Lindstrom et al 1981) and recently Larsson (1989) suggested a structure of such cubic phase dispersions; in these, the surface layer was proposed to be a lamellar phase, which immediately distinguishes such dispersions from the particles whose surface phase is L3 phase disclosed herein--the particles in this embodiment of the present invention are isotropic throughout, whereas those discussed by Larsson (1989) contain anisotropic, birefringent regions which are easily detected in the polarizing microscope. The only exception is particles, described in the present disclosure, which are surrounded by a lipid structure which is crystalline, not liquid crystalline as in the lamellar phase. Regarding the case of dispersions of reversed liquid crystalline phases by the use of a lamellar liquid crystalline phase as a dispersable phase the novel fragmentation technique according to the invention can be used.
2.4. Phase behavior in lipid-water based systems and the determination of cubic phases
A "lipid" is, in a broad view, defined as any molecule containing a substantial part of hydrocarbon. However, only those lipids that contain a hydrophilic polar part can give rise to liquid crystals by interactions with water. The basis for lipid lyotropic (and thermotropic) mesomorphism, and the formation of lipid assemblies, is the duality in solubility resulting from the presence of apolar (hydrophobic) and polar (generally hydrophilic) regions of the surfactant molecule--that is, its amphiphilicity (or amphipaticity). Amphiphilic lipids can be classified according to their interactions with water into nonpolar and polar (Small 1986). Where applicable within this disclosure we are concerned with lipids or lipid-like amphiphiles that exhibit mesomorphism and are thus classified as polar, insoluble and swelling amphiphiles. If nothing else is said we use the terminology introduced by Luzzati and associates (see Mariani et al. 1988, and ref. therein).
The principal techniques for studying the different phase structures are polarizing microscopy, X-ray diffraction, nuclear magnetic resonance (NMR) spectroscopy and electron microscopy techniques. Other techniques, as differential scanning calorimetry (DSC) and rheology can be used to give complementary information. Unambiguously phase determinations of the phases constituting the interior as well as the exterior is a prerequisite in order to classify dispersions according to the current invention. Preliminary phase behavior is usually carried out by texture analysis between crossed polarizers and more detailed in a polarizing microscope (Rosevear 1968). X-ray diffraction techniques are the obvious methods to deduce the symmetry of liquid crystals. The characterization of lipid mesophases by diffraction (Luzzati 1968) is based firstly on symmetry and the interpretation is normally based on treating the diffraction photographs as powder patterns. The long-range order of the assemblies in either one, two, or three dimensions, give rise to reflections which are converted to interplanar spacings. It is only with X-ray diffraction studies phase assignments can be regarded as unambiguous.
2.4.1. Phase diagrams in lipid-water systems
Fontell (1990) gives a comprehensive and systematic reveiw on cubic phase forming lipids and lipid-like surfactants and the occurrence of the cubic phases in the phase diagram and their relation to other phases. The information obtained from the structure of the neighboring phases can often be valuable for the identification of a cubic phase. The fact that a mesophase, such as the cubic or hexagonal phase, is in equilibrium with excess of water, is itself a strong indication that the structure is of the reversed, type II topology.
In the context of this invention, two examples of lipid-water based systems have been investigated with the objective of mapping the underlying phase behavior so as to understand and develop the techniques disclosed herein regarding the fragmentation process: Commercially available products have been used throughout this study, and it is important to note that these are generally not single-component products. We first discuss the binary phase diagram of the glycerol monooleate (GMO)-water system. The GMO has been obtained through molecular distillation of pine-needle oil (Grinsted, Denmark), and has a monoglyceride content of &gt;98%, of which 92.3% is monoolein (MO) (MO refers to the pure monoolein, while GMO refers to a monoolein rich monoglyceride blend). Many phase diagrams have been reported involving cubic phases of monoglycerides (Lutton (1965), Larsson et al. 1978, Krog and Larsson 1983, Larsson 1989, Krog 1990). In addition to the pure lipids monoolein, monoelaidin, monolinolein, monoarachidin, and monolinolein (Lutton 1965, Larsson et al. 1978, Hyde et al. 1984, Caffrey 1989), several blend qualities of monoacylglycerides are well characterized and known to form cubic phases in equilibrium with water (Larsson and Krog 1983, Krog 1990). Significantly, these blends are available at low production costs, typically less than $2 per pound.
Monoacylglycerides are often used in cosmetic products (Cosmetic Ingredient Review expert panel 1986), food industry (Krogh 1990) and pharmaceutics (Martindale the extra pharmacopoeia 1982), and are generally recognized as safe (GRAS) substances and as indirect food additives for human consumption without restrictions as to their concentrations. Federal regulations allow the use of monoglycerides, blends thereof, and blends of mono- and diglycerides as prior-sanctioned food ingredients and as both indirect and direct food additives. Furthermore, the metabolic fate of monoglycerides (and glycerides in general) is well documented in the human body. In the cosmetic industry monoglycerides and blends thereof, especially monoolein, are used as emulsifiers and thickening agents and recognized as safe cosmetic ingredients at concentrations up to 5% (Cosmetic Ingredient Review expert panel 1986).
The fact that there exists cubic phases in equilibrium with excess of water in the above mentioned monoglyceride systems is a strong indication that the cubic phase is of the reversed, type II topology. This has been verified by self-diffusion NMR (Lindblom et al. 1979). It should be pointed out that several systems which form cubic phases of the reversed type exhibit cubic mesomorphism, i.e. the appearance of a sequence of distinguishable cubic phases with different physical appearance, as well as exhibiting different lattice characteristics. The phase behavior of the present GMO-water system was; found to be very similar to that of MO-water reported by Hyde et al. (1984) (Engstrom and Engstrom 1992). The Q.sup.224 was found to be the cubic phase which coexists with excess of water.
The second lipid-water system used is the ternary system of GMO-soybean lecithin (SPC)-water. SPC is a pure phosphatidylcholine with the trade name Epikuron 200 which is well-characterized (Bergenstahl and Fontell 1983). It shares the general features of the phase diagram for MO-dioleoyl phosphatidylcholine-heavy water system reported by Gutman et al (1984). The existence of three cubic phases within the cubic region is experimentally verified by X-ray diffraction, as was the coexistence of cubic phases with excess of water.
2.4.2. Phase behavior and phase diagrams in lipid-protein-water systems
The phase properties in lipid-protein-water mixtures is a relatively unexplored area of research. Most of the studies have been reported by the Groups of Gulik-Krzywicki, Luzzati and colleagues (cf. Mariani et al. 1988, Gulik-Krzywicki 1975), by De Kruijff and coworkers (cf. Killian and De Kruijff 1986) and by Larsson and coworkers (cf. Ericsson et al. 1983, Ericsson 1986). Most studies address the behavior in diluted systems and often deal with the stability of the lamellar phase vs. the reversed hexagonal phase. The induction of non-lamellar phases is well established-for quite many systems. Some works address the phase properties vs. the activity of membrane bound enzymes, and it has been possible in some works to establish a correlation between an increased enzyme activity and isotropic movement of the lipid matrix. In the field of enzyme catalysis in microemulsions, some studies deal with the phase behavior; however, few works present phase diagrams.
That the cubic phases in the monoolein (MO)-water system could host quite large amounts of various substances, included proteins, had been known for many years (cf. Lindblom et al. 1979). The phase diagram of MO-lysozyme-water displays the general features of MO-protein-water systems, in cases where the protein is located in the aqueous labyrinths of the cubic phase (Ericsson et al. 1983). Ericsson (1986) reported a considerable number of proteins which can be incorporated within the MO-water cubic phase.
The second system which has been investigates in considerable detail is the MO-cytochrome c-water system reported by Luzzati and coworkers (Mariani et al. 1988), and it exhibits the general features found in the MO-lysozyme-water system. However, it also shows some features which necessary must arise from the protein; noteworthy is the existence of a chiral, non-centrosymmetric cubic phase, with space group 212. These aqueous MO-protein systems all exhibit at least one cubic phase which fulfils the criteria for constituting the interior phase of the particles according to the present invention.
2.5 Structure of the interior phases
The interior of the particles according to the invention consists of reversed lyotropic liquid crystalline phases, chosen from the group of reversed cubic liquid crystalline phases, reversed intermediate liquid crystalline phase, and reversed hexagonal liquid crystalline phase, or L3 phase, or a combination thereof. These phases are all well characterized and well established in the field of polymorphism of lipids and surfactants.
2.5.1. Structure of the cubic and hexagonal phases
Several reviews are available where cubic phases are discussed; see e.g. Luzzati (1968), Fontell (1974, 1978, 1981), Ekwall (1975), Tiddy (1980) and Luzzati et al. (1986). In recent years several surveys devoted to cubic phases have appeared. Luzzati and associates (Mariani et al. 1988) (see also Luzzati et al. 1987) give a detailed crystallographic description of the current situation with regard to the structure of the six cubic phases observed so far. Lindblom and Rilfors (1988) have reviewed the occurrence and biological implications of cubic phases formed by membrane lipids, and Larsson (1989) has reviewed the latest developments in the study of cubic lipid-water phases. A comprehensive review of the occurrence of cubic phases in literature phase diagrams was recently presented by Fontell (1990).
A general classification of the cubic phases is still not available. However, in the case of bilayer-bicontinuous cubic phases in binary systems they can be classified according to their interfacial mean curvature as "normal" (type I) or reversed (type II) cubic phases. Type cubic phases are those whose mean curvature at the apolar/polar interface is toward the apolar regions. Contrarily, type II or reversed cubic phases are those whose interface is towards the polar regions. In connection with the invention we are only concerned with cubic phases of type II, i.e. reversed.
Regarding the structure of the hexagonal phase it consists of hexagonally arranged rods of water (solvent) surrounded by a monolayer of amphiphile (see e.g. Seddon 1990, for a review).
2.5.2. Structure of the L3 phase
The microstructures of the L3 phases referred to are similar to those frequently found in surfactant-water systems (Benton et al. 1983, Porte et al. 1988, Gazeau et al. 1989, Anderson et al. 1989, Strey et al. 1990a, Strey et al. 1990b, Milner et al. 1990). The acquiescent L3 phase is isotropic. However, one striking and characteristic feature is that it shows extended flow birefringence. Other characteristics include long equilibration times and, at least relative to the amphiphile concentration, high viscosity. The structure is generally believed to be built up of multiply-connected bilayer forming a bicontinuous structure of high connectivity, and it may be regarded as a disordered counterpart to the cubic phases (Anderson et al. 1989), possessing similar topological connectivity and a local bilayer structure, but lacking long-range order.
2.6. Structure of the surface or dispersable phases
The structure of the L3 phase when used as the dispersable or fragmenting phase is exactly as described in 2.5.2. It should be pointed out that one bilayer of an L3 phase can not readily be distinguished from a lamella of a diluted lamellar phase. Similarly, it has been pointed out that the L3 phase may in certain systems exhibit metastability (Dubois and Zemb 1991) in which a transformation of the L3 phase to a lamellar phase was observed after 3 weeks of equilibration time. The lamellar structures, including lamellar phases with: disordered chains, untilted ordered or gel, and tilted gel, used as the dispersable phases are described by Luzzati (1968).